THE EFFECTS OF ETHYLENE GLYCOL VAPOR ON THE CONCENTRATION OF
CHLORPHYLL A AND B DURING RIPENING PROCESS OF BABY BANANA (Musa).
Noora Mosafaei Shirazi, Samouel Hanna and Pedram Khonsari. Department of Biological
Sciences, Saddleback College, Mission Viejo, CA, 92692.
During this experiment, individual bananas were taken from a green banana bunch and
placed into 3 groups (n=5). One group was exposed to 50 µL water, another to 50 µL
ethylene glycol and a reference group was designated. As banana ripening cycle involves
biosynthesis of ethylene, bananas in ethylene glycol group are expected to ripen more
during the 7 days of experiment. The peels of the reference banana group were removed,
weighted, and their juice was extracted, then spectrophotometric data was obtained. This
procedure was repeated for other groups. The mean of the total concentration of
chlorophyll a and b was calculated for all groups. Mean absorbance level ± SEM (4.22 ±
1.49, 2.09 ± 0.47, 0.73 ± 0.05, ANOVA p-value = 0.053). Comparing to the reference group,
chlorophyll a and b concentrations decreased by a factor of 2 in the water vapor group and
factor of 4 in the ethylene glycol group. No significant statistical difference existed. For
carotenoid concentrations ANOVA was performed (p-value = 0.0002), and the post-hoc test
showed no significant statistical difference among any of the groups.
Introduction
Many physical changes of fruits are believed to be hormone triggered processes that involves
biochemical cycles which induce a response on the cellular level. Banana ripening is a
physiological event that affects different physical properties. In terms of the life cycle of
bananas, ripening is a stage that allows for both the propagation of seeds and the production of
more fruits from the flowering part of the plant (Liu et al., 1999). Factors influencing banana
ripening are important in the world market since banana, as a food source, plays a significant part
in the socio-economic lives of millions of people in the developing world, its efficient and
healthy production is always required (Aquil et al., 2012). Initiation of ethylene cycle in banana
causes bananas to ripen for that matter; therefore, in the course of this experiment the group of
baby bananas exposed to ethylene glycol vapor is expected to ripen more during the 7 days of the
experiment. As the banana ripens the concentration of chlorophyll a and b decreases causes the
green color of the peel to change to yellow as more carotenoids are unmasked. Therefore, in this
experiment, the quantitative measure of banana ripening is the decrease in the concentration of
chlorgphyll a and b.
Materials and Methods
One bunch of green baby banana (Musa) was bought from a local grocery store. During this
experiment, individual bananas were taken from a green banana bunch and placed into 3 groups
(n=5). One group was exposed to 50 µL water, another to 50 µL ethylene glycol and a reference
group was designated. At the first day of this experiment, the peels of the reference bananas were
removed, weighted, and their juice was extracted by crushing the peels with a juicer and
adding15 mL acetone to the peel. After 10 minutes the liquid was poured in to a test tube. It was
centrifuged for 5 min, then the liquid portion was transferred to another test tube for
spectrophotometric readings. Concentration of chlorophyll a and b as well as carotenoids were
calculated by using concentration formulas (Boyer,1990 ).
The bananas of water vapor group and ethylene glycol group were both placed in a small
specimen jar, 50 µl of water was poured on a small patch and placed next to each banana in each
jars. Also, 50 µl of ethylene glycol was poured on each patch and placed next to each banana in
ethylene glycol group. The opening of the jar will be sealed by rubber stopper number 13 to
prevent the vapors from exiting the jars. Both groups were placed in Saddleback College stock
room. The bananas in both groups were photographed every day and Banana Ripeness Chart will
be used as a reference for staging the ripening process (UC Davis Postharvest Technology
Maintaining Produce Quality and Safety, 2013).
At the end of 7th day of experiment, the bananas in both ethylene glycol group and water vapor
groups were peeled. And the same procedure of juice extraction was repeated and
spectrophotometric data was obtained. Concentration of chlorophyll a and b as well as
carotenoids were calculated by using the same concentration formulas.
Ca = 11.24A661.6 - 2.04-A644.8
Cb = 20.13A644.8 - 4.19A661.6
Ca+b = 7.05A661.6 + 18.09A644.8
Cx+c = (1000A470 - 1.90Ca - 63.14Cb)/214
where: Ca = concentration of chlorophyll a in micrograms per milliliter of plant extract solution
(µg/ml), Cb = concentration of chlorophyll b in µg/ml, Ca+b = concentration of total chlorophyll in p,g/ml, Cx+c = concentration of total carotenoids (Boyer, 1990).
Results
Reference
Water Vapor
Ethylene glycol
µg/ml
µg/ml
µg/ml
Cx+c
2.130223791
2.257797911
1.273085952
Cx+c
2.455843065
1.577404463
1.273085952
Cx+c
4.082144584
1.836880631
1.014713605
Cx+c
3.994501374
1.461752656
1.247784053
Cx+c
3.774739417
1.869735372
1.014810173
Figure 1. Table containing the concentration of carotenoids in bananas in the reference group,
water vapor group and ethylene glycol group.
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1
5
16.43745223
3.28749
0.849914
Column 2
5
9.003571033
1.800714 0.09494
Column 3
5
5.823479736
1.164696 0.01884
ANOVA
Source of
Variation
SS
df
MS
Between
Groups
11.86879883
2
5.934399 18.47392 0.000217 3.885294
Within Groups
3.854775175
12
0.321231
Total
15.72357401
14
Comparison
Mean1 - Mean2
F
P-value
95% CI of difference
1: Reference vs. Water Vapor
+ 1.486776239
- 10.297749407 to + 13.271301885
2: Reference vs. Ethylene glycol
+ 2.122794499
- 9.661731147 to + 13.907320145
3: Water Vapor vs. Ethylene glycol
+ 0.636018260
- 11.148507386 to + 12.420543906
Comparison
Significant? (P <0.05?)
t
1: Reference vs. Water Vapor
No
0.965
2: Reference vs. Ethylene glycol
No
1.378
3: Water Vapor vs. Ethylene glycol
No
0.413
Comparison
Mean 1
Mean 2
N1
N2
1: Reference vs. Water Vapor
+ 3.287490446
+ 1.800714207
5
5
2: Reference vs. EG
+ 3.287490446
+ 1.164695947
5
5
3: Water Vapor vs. Ethylene glycol
+ 1.800714207
+ 1.164695947
5
5
F crit
Mean absorbance level ± SEM (4.22 ± 1.49, 2.09 ± 0.47, 0.73 ± 0.05, ANOVA p-value = 0.053).
Comparing to the reference group, chlorophyll a and b concentrations decreased by a factor of 2
in the water vapor group and factor of 4 in the ethylene glycol group. No significant statistical
difference existed. For carotenoid concentrations ANOVA was performed (p-value = 0.0002),
and the post-hoc test showed no significant statistical difference among any of the groups.
Reference Group µg/ml
Water Vapor Group µg/ml Ethylene glycol Group µg/ml
C a+b
2.2368
2.18439
0.84799
C a+b
3.09648
1.24317
0.84799
C a+b
10.1244
1.73985
0.62997
C a+b
2.28402
3.8697
0.75857
C a+b
3.36504
1.44336
0.60785
Figure 2. Total chlorophyll a and b concentration of reference group, water vapor group and
ethylene glycol group.
Anova: Single Factor
SUMMARY
Groups
Count
Sum
Average
Variance
Column 1
5
21.10677
4.221354
11.13416
Column 2
5
10.48047
2.096094
1.108481
Column 3
5
3.69237
0.738474
0.013307
ANOVA
Source of
Variation
SS
Between
Groups
df
MS
F
P-value
F crit
30.81719 2
15.4086
3.771702 0.05359 3.885294
Within Groups
49.02379 12
4.085316
Total
79.84098 14
Discussion
During the course of experiment different chemical reactions contribute to the ripening of
bananas. This type of ripening process is present in many fruits; in fact “ Apple, avocado,
banana, and peer fruits exhibit a marked increase in respiration rate, the climacteric during
ripening. It is during this period of accelerated respiratory activity that fruits soften, lose
chlorophyll, develop flavor and aroma” (Frenkel et al., 1968).The banana ripening cycle (Yang
Cycle) involves biosynthesis of ethylene which causes the yellowing of banana peel and
softening of the pulp. When bananas were bought they were un-ripen- at the third stage of the
ripening. In this stage the shades of green were still present toward the tip of the bananas and the
lower half of the fruit and banana body were hard. During the course of ripening different
physical changes indicate the onset of ripening cycle in the fruit. “Bananas are climacteric fruit
in which ripening is regulated by ethylene. Once initiated, ripening involves numerous changes
including the conversion of starch to sugars, alterations in fruit texture and the synthesis of
volatile compounds.” (Drury et al., 1999). Although majority of clues concerning the ripening of
banana are represented in the appearance of both peel and pulp. Some of these changes vary
between the peel and pulp. “The peel tissue in banana appears to share many ripening related
events with the pulp including softening and starch degradation, but the presence and
degradation of chlorophyll (Chl) are unique to the peel” (Drury et al., 1999).
Citation
Aquil, B., Jan, A.T., Sarin, N.B. and Haq, Q.R. (2012). Micropropagation and genetic
transformation of banana for crop improvement and sustainable agriculture. Journal of Crop
Science. 3(2): 64-77.
Boyer, R.F. (1990). Isolation and spectrophotometric characterization of photosynthetic
pigments. Biochemical Education. 18(4): 203-206.
Liu, X., Shiomi, A., Nakatsuka, A., Kubo, Yasutaka., Nakamura, R. and Inaba, A. (1999).
Characterization of ethylene biosynthesis associated with ripening in banana fruit. Plant
Physiology. 121: 1257-1265.
UC Davis Postharvest Technology Maintaining Produce Quality and Safety
[Internet].c2013.California: Regents of the University of California; [cited 2013 Sep 29].
Available from:
http://postharvest.ucdavis.edu/PFfruits/BananaPhotos/?repository=29280&a=83211
Xiao, Y., Chen, J., Kuang, J., Shan, W., Xie, H., Jiang, Y. and Lu, W. (2013). Banana ethylene
response factors are involved in fruit ripening through their interactions with ethylene
biosynthesis genes. Journal of Experimental Botany Advance Access. 1: 1-12.